This invention relates to novel mutant enzymes or enzyme variants useful in formulating detergent compositions and exhibiting improved storage stability while retaining or improving their wash performance; cleaning and detergent compositions containing said enzymes; mutated genes coding for the expression of said enzymes when inserted into a suitable host cell or organism; and such host cells transformed therewith and capable of expressing said enzyme variants.
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important are proteases.
Although proteases have been used in the detergent industry for more than 30 years, much remains unknown as to details of how these enzymes interact with substrates and/other substances present in e.g. detergent compositions. Some factors related to specific residues and influencing certain properties, such as oxidative and thermal stability in general have been elucidated, but much remains to be found out. Also, it is still not exactly known which physical or chemical characteristics are responsible for a good washing performance or stability of a protease in a specific detergent composition.
The currently used proteases have for the most part been found by isolating proteases from nature and testing them in detergent formulations.
An increasing number of commercially used protease are protein engineered variants of the corresponding naturally occurring wild type protease, e.g. DURAZYM(copyright) (Novo Nordisk A/S), RELASE(copyright) (Novo Nordisk A/S), MAXAPEM(copyright) (Gist-Brocades N.V.), PURAFECT(copyright) (Genencor International, Inc.).
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3). Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral, or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase and usually referred to as subtilisin. Secretion of these proteases has been linked to the bacterial growth cycle, with greatest expression of protease during the stationary phase, when sporulation also occurs. Joliffe et al. (1980) J. Bacteriol 141 1199-1208, have suggested that Bacillus proteases finction in cell wall turnover.
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilisins have been identified, and the amino acid sequence of a number of subtilisins have been determined. These include more than six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN"", subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus, and mesentericopeptidase (Kurihara et al. (1972) J. Biol. Chem. 247 5629-5631; Wells et al. (1983) Nucleic Acids Res. 11 7911-7925; Stahl and Ferrari (1984) J. Bacteriol. 159 811-819, Jacobs et al. (1985) Nucl. Acids Res. 13 8913-8926; Nedkov et al. (1985) Biol. Chem. Hoppe-Seyler 366 421-430, Svendsen et al. (1986) FEBS Lett. 196 228-232), one subtilisin from an actinomycetales, thermitase from Thermoactinomyces vulgaris (Meloun et al. (1985) FEBS Lett. 198 195-200), and one fungal subtilisin, proteinase K from Tritirachium album (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler 366 584-492). for further reference Table I from Siezen et al. has been reproduced below.
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisins have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors, and define the structural consequences for natural variation (Kraut (1977) Ann. Rev. Biochem. 46 331-358).
In the context of this application substrate should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Also the expression xe2x80x9cproductxe2x80x9d should in the context of this invention be interpreted to include the products of a hydrolysis reaction involving a subtilisin protease. A product may be the substrate in a subsequent hydrolysis reaction.
One subgroup of the subtilases, I-S1, comprises the xe2x80x9cclassicalxe2x80x9d subtilisins, such as subtilisin 168, subtilisin BPN"", subtilisin Carlsberg (ALCALASE(copyright), Novo Nordisk A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognised by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL(copyright), Gist-Brocades N.V., subtilisin 309 (SAVINASE(copyright), Novo Nordisk A/S), subtilisin 147 (ESPERASE(copyright), Novo Nordisk A/S), and alkaline elastase YaB.
In the context of this invention, a subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilisin protease is produced when expressed in a suitable host.
Random and site-directed mutations of the subtilase gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilase""s catalytic activity, substrate specificity, tertiary structure, etc. (Wells et al. (1987) Proc. Natl. Acad. Sci. USA. 84; 1219-1223; Wells et al. (1986) Phil. Trans. R. Soc. Lond.A. 317 415-423; Hwang and Warshel (1987) Biochem. 26 2669-2673; Rao et al., (1987) Nature 328 551-554.
More recent publications covering this area are Carter et al. (1989) Proteins 6 240-248 relating to design of variants that cleave a specific target sequence in a substrate (positions 24 and 64); Graycar et al. (1992) Annals of the New York Academy of Sciences 672 71-79 discussing a number of previously published results; and Takagi (1993) Int. J. Biochem. 25 307-312 also reviewing previous results.
Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in the following patent applications and patents:
EP 130 756 (Genentech) (corresponding to U.S. Reissue Patent No. 34,606 (Genencor)) relating to site specific or randomly generated mutations in xe2x80x9ccarbonyl hydrolasesxe2x80x9d and subsequent screening of the mutated enzymes for various properties, such as kcat/Km ratio, pH-activity profile, and oxidation stability. This publication reveals that site-specific mutation is feasible, and that mutation of subtilisin BPN"" in certain specified positions, i.e. xe2x88x921Tyr, 32Asp, 155Asn, 104Tyr, 222Met, 166Gly, 64His, 169Gly, 189Phe, 33Ser, 221Ser, 217Tyr, 156Glu or 152Ala, provide for enzymes exhibiting altered properties. Since these positions all except position xe2x88x921 were known to be involved in the functioning of the enzyme prior to the filing of the application, and therefore evident to select, this application does not contribute much to solving the problem of deciding where to introduce mutations in order to obtain enzymes with desired properties.
EP 214 435 (Henkel) relating to cloning and expression of subtilisin Carlsberg and two mutants thereof. In this application no reason for mutation of 158Asp to 158Ser and 161Ser to 161Asp is provided.
In International patent publication No. WO 87/04461 (Amgen) it is proposed to reduce the number of Asn-Gly sequences present in the parent enzyme in order to obtain mutated enzymes exhibiting improved pH and heat stabilities, in the application emphasis is put on removing, mutating, or modifying the 109Asn and the 218Asn residues in subtilisin BPN"". No examples are provided for any deletions or for modifying the Gly-residues.
International patent publication No. WO 87/05050 (Genex) discloses random mutation and subsequent screening of a large number of mutants of subtilisin BPN"" for improved properties. In the application mutations are described in positions 218Asn, 131Gly, 254Thr, 166Gly, 116Ala, 188Ser, 126Leu, and 53Ser.
In EP 251 446 (Genencor) it is described how homology considerations at both primary and tertiary structural levels may be applied to identify equivalent amino acid residues whether conserved or not. This information together with the inventors knowledge of the tertiary structure of subtilisin BPN"" lead the inventors to select a number of positions susceptible to mutation with an expectation of obtaining mutants with altered properties. The positions so identified are: 124Met, 222Met, 104Tyr, 152Ala, 156Glu, 166Gly, 169Gly, 189Phe, 217Tyr. Also 155Asn, 21Tyr, 22Thr, 24Ser, 32Asp, 33Ser, 36Asp, 46Gly, 48Ala, 49Ser, 50Met, 77Asn, 87Ser, 94Lys, 95Val, 96Leu, 107Ile, 110Gly, 170Lys, 171Tyr, 172Pro, 197Asp, 199Met, 204Ser, 213Lys, and 221Ser, which positions are identified as being expected to influence various properties of the enzyme. Also, a number of mutations are exemplified to support these suggestions. In addition to single mutations in these positions the inventors also performed a number of multiple mutations. Further the inventors identify 215Gly, 67His, 126Leu, 135Leu, and amino acid residues within the segments 97-103, 126-129, 213-215, and 152-172 as having interest, but mutations in any of these positions are not exemplified.
Especially of interest for the purpose of the present invention the inventors of EP 251 446 suggest to substitute 170Lys (in subtilisin BPN"", type I-S1), specifically they suggest to introduce Glu or Arg for the original Lys. It appears that the Glu variant was produced and it was found that it was highly susceptible to autolytic degradation (cf. pages 48, 121, 123 (Table XXI includes an obvious error, but indicates a reduction in autolysis half-time from 86 to 13 minutes) and FIG. 32).
EP 260 105 (Genencor) describes modification of certain properties in enzymes containing a catalytic triad by selecting an amino acid residue within about 15 xc3x85 from the catalytic triad and replace the selected amino acid residue with another residue. Enzymes of the subtilase type described in the present specification are specifically mentioned as belonging to the class of enzymes containing a catalytic triad. In subtilisins positions 222 and 217 are indicated as preferred positions for replacement.
Also, it has been shown by Thomas, Russell, and Fersht (1985) Nature 318 375-376 that exchange of 99Asp into 99Ser in subtilisin BPN"" changes the pH dependency of the enzyme.
In a subsequent article (1987) J. Mol. Biol. 193 803-813, the same authors also discuss the substitution of 156Ser in place of 156Glu.
Both these mutations are within a distance of about 15 xc3x85 from the active 64His.
In Nature 328 496-500 (1987) Russel and Fersht discuss the results of their experiments and present rules for changing pH-activity profiles by mutating an enzyme to obtain changes in surface charge.
WO 88/08028 (Genex) and WO 88/08033 (Amgen) both relate to modifications of amino acid residues in the calcium binding sites of subtilisin BPN"". The enzyme is said to be stabilized by substituting more negatively charged residues for the original ones.
In WO 89/06279 (Novo Nordisk A/S) position 170 is indicated as interesting and it is suggested to replace the existing residue with Tyr. However, no data are given in respect of such a variant. In WO 91/00345 (Novo Nordisk A/S) the same suggestion is made, and it is shown that the Tyr variant of position 170 in subtilisin 309 (type I-S2) exhibits an improved wash performance in detergents at a pH of about 8 (variant S003 in Tables III, IV, V, VI, VIII, X). The same substitution in combination with other substitutions in other positions also indicates an improved wash performance(S004, S011-S014, S022-S024, S019, S020, S203, S225, S227 in the same Table and Table VII) all in accordance with the generic concept of said application.
In EP 525 610 (Solvay) it is suggested to improve the stability of the enzyme (a type I-S2 subtilase closely related to subtilisin PB92) towards ionic tensides by decreasing the hydrophobicity in certain surface regions thereof. It is consequently suggested to substitute Gln for the Arg in position 164 (170 if using BPN"" numbering). No variants comprising this substitution are disclosed in the application.
In WO 94/02618 (Gist-Brocades N.V.) a number of position 164 (170 if using BPN"" numbering) variants of the I-S2 type subtilisin PB92 are described. Examples are provided showing substitution of Met, Val, Tyr, Ile, for the original Arg. Wash performance testing in powder detergents of the variants indicates a slight improvement. Especially for the Ile variant wash performance tests on cacao an improvement of about 20-30% is indicated. No stability data are provided.
In WO 95/30011, WO 95/30010, and WO 95/29979 (Procter and Gamble Company) describe 6 regions, especially position 199-220 (BPN"" numbering), in both Subtilisin BPN"" and subtilisin 309, which are designed to change (i.e. decrease) the adsorption of the enzyme to surface-bound soils. It is suggested that decreased adsorption by an enzyme to a substrate results in better detergent cleaning performance. No specific detergent wash performance data are provided for the suggested variants.
WO 95/27049 (Solvay S.A.) describes a subtilisin 309 type protease with following mutations: N43R+N116R+N117R (BPN"" numbering). Data indicate the corresponding variant is having improved stability, compared to wildtype.
Proteases such as subtilisins have found much utility in industry, particularly in detergent formulations, as they are useful for removing proteinaceous stains.
At present at least the following proteases are known to be commercially available and many of them are marketed in large quantities in many countries of the world.
Subtilisin BPN"" or Novo, available from e.g. Sigma, St. Louis, U.S.A.
Subtilisin Carlsberg, marketed by Novo Nordisk A/S (Denmark) as ALCALASE(copyright) and by Gist-Brocades N.V. (Holland) as MAXATASE(copyright);
Both of these belong to subtilase subgroup I-S1
Among the subtilase sub-group I-S2 the following are known to be marketed.
A Bacillus lentus subtilisin, subtilisin 309, marketed by Novo Nordisk A/S (Denmark) as SAVINASE(copyright). A protein engineered variant of this enzyme is marketed as DURAZYM(copyright).
Enzymes closely resembling SAVINASE(copyright), such as subtilisin PB92, MAXACAL(copyright) marketed by Gist-Brocades N.V. (a protein engineered variant of this enzyme is marketed as MAXAPE(copyright)), OPTICLEAN(copyright) marketed by Solvay et Cie. and PURAFECT(copyright) marketed by Genencor International.
A Bacillus lentus subtilisin, subtilisin 147, marketed by Novo Nordisk A/S (Denmark) as ESPERASE(copyright);
To be effective, however, such enzymes must not only exhibit activity under washing conditions, but must also be compatible with other detergent components during detergent production and storage.
For example, subtilisins may be used in combination with other enzymes active against other substrates, and the selected subtilisin should possess stability towards such enzymes, and also the selected subtilisin preferably should not catalyse degradation of the other enzymes. Also, the chosen subtilisin should be resistant to the action from other components in the detergent formulation, such as bleaching agents, oxidizing agents, etc., in particular an enzyme to be used in a detergent formulation should be stable with respect to the oxidizing power, calcium binding properties, and pH conditions rendered by the non-enzymatic components in the detergent during storage and in the wash liquor during wash.
The ability of an enzyme to catalyze the degradation of various naturally occurring substrates present on the objects to be cleaned during e.g. wash is often referred to as its washing ability, washability, detergency, or wash performance. Throughout this application the term wash performance will be used to encompass this property.
The ability of an enzyme to remain active in the presence of other components of a detergent composition prior to being put to use (normally by adding water in the washing process) is usually referred to as storage stability or shelf life. It is often measured as half-life, txc2xd. We will use the expression storage stability for this property throughout this application to encompass this property.
Naturally occurring subtilisins have been found to possess properties which are highly variable in relation to their washing power or ability under variations in parameters such as pH. Several of the above marketed detergent proteases, indeed, have a better performance than those marketed about 20 years ago, but for optimal performance each enzyme has its own specific conditions regarding formulation and wash conditions, e.g. pH, temperature, ionic strength (=I), active system (tensides, surfactants, bleaching agent, etc.), builders, etc.
As a consequence it is found that an enzyme possessing desirable properties at low pH and low I may be less attractive at more alkaline conditions and high I, or an enzyme exhibiting fine properties at high pH and high I may be less attractive at low pH, low I conditions.
Also, it has been found that the storage stability differs between the enzymes, but it has further been found that a specific enzyme exhibits large variations in storage stability in respect of different detergent formulations, dependent upon a number of parameters, such as pH, pI, bleach system, tensides, etc., and upon the physical state of the detergent compositions, which may be in powder, dust, or liquid form. Furthermore it may be concentrated or dilute.
The advent and development of recombinant DNA techniques has had a profound influence in the field of protein chemistry.
Through the application of this technology it is possible now to construct enzymes having desired amino acid sequences, and as indicated above a fair amount of research has been devoted to designing subtilisins with altered properties.
Among the proposals the technique of producing and screening a large number of mutated enzymes as described in EP 130 756 (Genentech) (U.S. Reissue Pat. No. 34,606 (Genencor)) and International patent publ. no. WO 87/05050 (Genex) correspond to a large extend to the classical method of isolating native enzymes, submit them to classical mutagenesis programs (using radiation or chemical mutagens) and screen them for their properties. The difference lies in that these methods are more efficient through the knowledge of the presence of a large number of variant enzymes substituted in a specific position.
A subtilisin enzyme typically comprises about 275 amino acid residues. Each residue is capable of being 1 out of 20 possible naturally occurring amino acids.
Therefore one very serious draw-back in that procedure is the very large number of mutations generated that have to be submitted to a number of preliminary screenings to determine their properties.
A procedure as outlined in these patent applications will consequently only be slightly better than the traditional random mutation procedures which have been known for years.
The other known techniques relate to changing specific properties, such as oxidation stability, thermal stability, Ca-stability, transesterification and hydrolysis rate (EP 260 105 (Genencor)), pH-activity profile (Thomas, Russell, and Fersht, supra), and substrate specificity (International patent publ. no. WO 88/07578 (Genentech)). None of these publications relates to changing either the wash performance of enzymes or their storage stability.
In International Patent Application no. PCT/DK 88/00002 (Novo Nordisk A/S) it is proposed to use the concept of homology comparison to determine which amino acid positions should be selected for mutation and which amino acids should be substituted in these positions in order to obtain a desired change in wash performance.
By using such a procedure the task of screening is reduced drastically, since the number of mutants generated is much smaller, but with that procedure it is only foreseen that enzymes exhibiting the combined useful properties of the parent enzyme and the enzyme used in the comparison may be obtained.
Thus, as indicated above no relationship has yet been identified between well defined properties of an enzyme such as those mentioned above and the wash performance and storage stability of an enzyme in various detergent compositions.
The problem seems to be that although much research has been directed at revealing the mechanism of enzyme activity, still only little is known about the factors in structure and amino acid residue combination that determine the properties, such as storage stability in detergents, of enzymes in relation to most of their characteristics, especially when the enzymes are present in complex mixtures.
Consequently there still exists a need for further improvement and tailoring of enzymes to detergent systems, as well as a better understanding of the mechanism of protease action and degradation in the practical use of cleaning or detergent compositions. Such an understanding could result in rules which may be applied for selecting mutations that with a reasonable degree of certainty will result in an enzyme exhibiting improved storage stability under specified conditions in a detergent composition.
It has now surprisingly been found that a subtilase variant having improved storage stability and/or improved performance in detergents, can be obtained by substituting one or more amino acid residues situated in, or in the vicinity of a hydrophobic domain of the parent subtilase for an amino acid residue more hydrophobic than the original residue, said hydrophobic domain comprising the residues corresponding to residues P129, P131, I165, Y167, Y171 of BLS309 (in BASBPN numbering), and said residues in the vicinity thereof comprises residues corresponding to the residues E136, G159, S164, R170, A194, and G195 of BLS309 (in BASBPN numbering), with the exception of the R170M, R170I and R170V variants of BABP92.
The present invention relates consequently in its first aspect to enzyme variants exhibiting improved stability and/or improved wash performance in detergent.
In its second aspect the invention relates to DNA constructs capable of expressing the enzymes of the first aspect, when inserted in a suitable manner into a host cell that subsequently is brought to express the subtilisin enzyme(s) of the first aspect.
In a third aspect the invention relates to the production of the subtilisin enzymes of the invention by inserting a DNA construct according to the second aspect into a suitable host, cultivating the host to express the desired subtilase enzyme, and recovering the enzyme product.
The invention relates, in part, but is not limited to, mutants of the genes expressing the subtilase sub-group I-S2 enzymes and the ensuing enzyme variants, as indicated above.
Other subtilase gene variants encompassed by the invention are such as those of the subtilase subgroup I-S1, e.g. Subtilisin BPN"", and Subtilisin Carlsberg genes and ensuing variant Subtilisin BPN"", Proteinase K, and Subtilisin Carlsberg enzymes, which exhibit improved stability in concentrated liquid detergents.
Still further subtilase gene variants encompassed by the invention are such as Proteinase K and other genes and ensuing variant Proteinase K, and other subtilase enzymes, which exhibit improved stability in concentrated liquid detergents.
Other examples of parent subtilase enzymes that can be modified in accordance with the invention are listed in Table I.
Further the invention relates to the use of the mutant enzymes in cleaning compositions and cleaning compositions comprising the mutant enzymes, especially detergent compositions comprising the mutant subtilisin enzymes. Specifically the invention relates to concentrated liquid detergent compositions comprising such enzyme variants.
In describing the various enzyme variants produced or contemplated according to the invention, the following nomenclatures have been adapted for ease of reference:
Original amino acid(s) position(s) substituted amino acid(s)
According to this the substitution of Glutamic acid for glycine in position 195 is designated as:
Gly 195 Glu or G195E a deletion of glycine in the same position is:
Gly 195* or G195* and insertion of an additional amino acid residue such as lysine is:
Gly 195 GlyLys or G195GK
Where a deletion in comparison with the sequence used for the numbering is indicated, an insertion in such a position is indicated as:
*36 Asp or *36D for insertion of an aspartic acid in position 36
Multiple mutations are separated by pluses, i.e.:
Arg 170 Tyr+Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
In describing the variants in this application and in the appended claims use is made of the alignment of various subtilases in Siezen et al., Supra. In other publications relating to subtilases other alignments or the numbering of specific enzymes have been used. It is a routine matter for the skilled person to establish the position of a specific residue in the numbering used here. Reference is also made to FIG. 1 showing an alignment of residues relevant for the present invention from a large number of subtilases. Reference is also made to Table I of WO 91/00345 showing an alignment of residues relevant for the present invention from a number of subtilases.
References to amino acid sequences (GenBank(copyright)/EMBL Data Bank accession numbers are shown in brackets):
ARB172 Kamekura and Seno, (1990) Biochem. Cell Biol. 68 352-359 (amino acid sequencing of mature protease residues 1-35; residue 14 not determined).
BSS168 Stahl and Ferrari, (1984) J. Bacteriol. 158, 411-418 (K01988). Yoshimoto, Oyama et al. (1488) J. Biochem. 103, 1060-1065 (the mature subtilisin from B. subtilis var. amylosacchariticus differs in having T130S and T162S). Svendsen, et al. (1986) FEBS Lett. 196, 228-232 (PIR A23624; amino acid sequencing; the mature alkaline mesentericopeptidase From B. mesentericus differs in having S85A, A88S, S89A, S183A and N259S).
BASBPN Wells, et al. (1983) Nucl. Acids Res. 11 7911-7925 (X00165). Vasantha et al., (1984) J. Bacteriol. 159 811-814(K02496).
BSSDY Nedkov et al. (1983) Hoppe-Seyler""s Z. Physiol. Chem. 364 1537-1540 (PIR A00969; amino acid sequencing).
BLSCAR Jacobs et al. (1985) Nucleic Acids Res. 13 8913-8926 (X03341). Smith et al. (1968) J. Biol. Chem. 243 2184-2191 (PIR A00968; amino acid sequencing; mature protease sequence differs in having T103S, P129A, S158N, N161S and S212N).
BLS147 Hastrup et al. (1989) PCT Patent Appl. WO 8906279. Pub. Jul. 13 1989. (Esperase(copyright) from B. lentus). Takami et al. (1990) Appl. Microbiol. Biotechnol., 33 519-523 (amino acid sequencing of mature alkaline protease residues 1-20 from Bacillus sp. no. AH-101; this sequence differs from BLS147 in having N11S).
BABP92 van der Laan et al. (1991) Appl. Environ. Microbiol. 57 901-909. (Maxacal(copyright)). Hastrup et al. (1989) PCT Patent Appl. WO 8906279. Pub. Jul. 13, 1989. (subtilisin 309). Savinase(copyright), from B. lentus differs only in having N87S). Godette et al. (1991) Abstracts 5th Protein Society Symposium, June 6, Baltimore: abstract M8 (a high-alkaline protease from B. lentus differs in having N87S, S99D, S101R, S103A, V104I and G159S).
BDSM48 Rettenmaier et al. (1990) PCT Patent Appl. WO 90/04022. Publ. Apr. 19, 1990.
BYSYAB Kaneko et al. (1989) J. Bacteriol. 171 5232-5236 (M28537).
BSEPR Sloma et al. (1988) J. Bacteriol. 170 5557-5563 (M22407). Bruckner (1990) Mol. Gen. Genet. 221 486-490 (X53307).
BSBPF Sloma et al. (1990) J. Bacteriol. 172 1470-1477 (M29035; corrected). Wu et al. (1990) J. Biol Chem. 265 6845-6850 (J05400; this sequence differs in having A169V and 586 less C-terminal residues due to a frameshift).
BSISP1 Koide et al. (1986) J. Bacteriol. 167 110-116 (M13760).
BSIA50 Strongin et al. (1978) J. Bacteriol. 133 1401-1411 (amino acid sequencing of mature protease residues 1-54; residues 3, 39, 40, 45, 46, 49 and 50 not determined).
BTFINI Chestukhina et al. (1985) Biokhimiya 50 1724-1730 (amino acid sequencing of mature protease residues 1-14 from B. thuringiensis variety israeliensis, and residues 1-16 and 223-243 from variety finitimus). Kunitate et al. (1989) Agric. Biol. Chem. 53 3251-3256 (amino acid sequencing of mature protease residues 6-20 from variety kurstaki. BTKURS).
BCESPR Chestukhina et al. (1985) Biokhimiya 50 1724-1730 (amino acid sequencing of mature residues 1-16 and 223-243).
NDAPII Tsujibo et al. (1990) Agric. Biol. Chem. 54 2177-2179 (amino acid sequencing of mature residues 1-26).
TVTHER Meloun et al. (1985) FEBS Lett. 183 195-200 (PIR A00973; amino acid sequencing of mature protease residues 1-274).
EFCYLA Segarra et al. (1991) Infect. Immun. 59 1239-1246.
SEEPIP Schnell et al. (1991) personal communication (Siezen et al. (supra)).
SPSCPA Chen et al. (1990) J. Biol. Chem. 265 3161-3167 (J05224).
DNEBPR Kortt et al. (1991) Abstracts 5th Protein Society Symposium, June 22-26, Baltimore, abstract S76.
LLSK11 Vos et al. (1989) J. Biol. Chem. 264 13579-13585 (J04962). Kok et al. (1988) Appl. Environ. Microbiol. 54 231-238 (M24767; the sequence from strain Wg2 differs in 44 positions, including 18 differences in the protease domain, and a deletion of residues 1617-1676). Kiwaki et al. (1989) Mol. Microbiol. 3 359-369 (X14130; the sequence from strain NCD0763 differs in 46 positions, including 22 in the protease domain, and a deletion of residues 1617-1676).
XCEXPR Liu et al. (1990) Mol. Gen. Genet. 220 433-440.
SMEXSP Yanagida et al. (1986) J. Bacteriol. 166 937-994 (M13469).
TAAQUA Terada et al. (1990) J. Biol. Chem. 265 6576-6581 (J054I4).
TRT41A McHale et al. (1990) Abstracts 5th Eur. Congr. Biotechn. Christiansen, Munck and Villadsen (eds), Munksgaard Int. Publishers, Copenhagen.
VAPROA Deane et al. (1989) Gene 76 281-288 (M25499).
SRESPD Lavrenova et al. (1984) Biochemistry USSR. 49 447-454 (amino acid sequencing of residues 1-23; residues 13, 18 and 19 not determined).
AVPRCA Maldener et al. (1991) Mol. Gen. Genet. 225 113-120 (the published sequence has 28 uncertain residues near position 200-210 due to a frameshift reading error).
TAPROK Gunkel and Gassen (1989) Eur. J. Biochem. 179 185-194 (X14688/XI4689). Jany et al. (1986) J. Biol. Chem. Hoppe-Seyler 367 87(PIR A24541; amino acid sequencing; mature protease differs in having S745G, SILST204-208DSL and VNLL264-267FNL).
TAPROR Samal et al. (1990) Mol. Microbiol. 4 1789-1792 (X56116).
TAPROT Samal et al. (1989) Gene 85 329-333.
AOALPR Tatsumi et al. (1989) Mol. Gen. Genet. 219 33-38. Cheevadhanarah et al. (1991) EMBL Data Library (X54726).
MPTHMY Gaucher and Stevenson (1976) Methods Enzymol. 45 415-433 (amino acid sequencing of residues 1-28, and hexapeptide LSGTSM with active site serine).
ACALPR Isogai et al. (1991) Agric. Biol. Chem. 55 471-477. Stepanov et al. (1986) Int. J. Biochem. 18 369-375 (amino acid sequencing of residues 1-27: the mature protease differs in having H13[1]Q, R13[2]N and S13[6]A).
KLKEX1 Tanguy-Rougeau, Wesolowski-Louvel and Fukuhara (1988) FEBS lett. 234 464-470 (X07038).
SCKEX2 Mizuno et al. (1988) Biochem. Biophys. Res. Commun. 156 246-254(M24201).
SCPRB1 Moehle et al. (1987) Mol. Cell. Biol. 7 4390-4399 (M18097).
YLXYPR2 Davidow et al. (1987) J. Bacteriol. 169 4621-4629 (M17741). Matoba et al. (1988) Mol. Cell Biol. 8 4904-4916 (M23353).
CEBL14 Peters and Rose (1991) The Worm Breeder""s Gazette 11 28.
DMFUR1 Roebroek et al. (1991) FEBS Lett. 289 133-137 (X59384).
DMFUR2 Roebroek et al. (1992) 267 17208-17215.
CMCUCU Kaneda et al. (1984) J. Biochem. 95 825-829 (amino acid sequencing of octapeptide NIISGTSM with active site serine).
HSFURI van den Ouweland et al. (1990) Nucl. Acids Res. 18 664 (X04329) (the sequence of mouse furin differs in 51 positions, including five in the catalytic domain: A15E, Y21F, S223F, A232V and N258[2]D). Misumi et al.(1990) Nucl. Acids Res. 18 6719 (X55660: the sequence of rat furin differs in 49 positions, including three in the catalytic domain: A15E, Y21F, H24R).
HSIPC2 Smeekens and Steiner (1990) J. Biol. Chem. 265 2997-3000 (J05252). Seidah et al. (1990) DNA Cell Biol. 9 415-424 (the sequence of mouse pituitary PC2 protease differs in 23 positions, including seven in the protease domain: I4F, S42[2]Y, E45D, N76S, D133E, V134L and G239[1]D).
MMPPC3 Smeekens et al. (1991) Proc. Natl. Acad. Sci. USA 88 340-344 (M58507). Seidah et al. (1990) DNA Cell Biol. 9 415-424 (M55668/M55669; partial sequence).
HSTPP Tomkinson and Jonsson (1991) Biochemistry 30 168-174 (J05299).